NAD+-dependent isocitrate dehydrogenase. Cloning, nucleotide sequence, and disruption of the IDH2 gene from Saccharomyces cerevisiae
1991
Cupp, J.R. | McAlister-Henn, L.
NAD+-dependent isocitrate dehydrogenase from Saccharomyces cerevisiae is composed of two nonidentical subunits, designated IDH1 (Mr approximately 40,000) and IDH2 (Mr approximately 39,000). We have isolated and characterized a yeast genomic clone containing the IDH2 gene. The amino acid sequence deduced from the gene indicates that IDH2 is synthesized as a precursor of 369 amino acids (Mr 39,694) and is processed upon mitochondrial import to yield a mature protein of 354 amino acids (Mr 37,755). Amino acid sequence comparison between S. cerevisiae IDH2 and S. cerevisiae NADP+-dependent isocitrate dehydrogenase shows no significant sequence identity, whereas comparison of IDH2 and Escherichia coli NADP+-dependent isocitrate dehydrogenase reveals a 33% sequence identity. To confirm the identity of the IDH2 gene and examine the relationship between IDH1 and IDH2, the IDH2 gene was disrupted by genomic replacement in a haploid yeast strain. The disruption strain expressed no detectable IDH2, as determined by Western blot analysis, and was found to lack NAD+-dependent isocitrate dehydrogenase activity, indicating that IDH2 is essential for a functional enzyme. Overexpression of IDH2, however, did not result in increased NAD+-dependent isocitrate dehydrogenase activity, suggesting that both IDH1 and IDH2 subunits are required for catalytic activity. The disruption strain was unable to utilize acetate as a carbon source and exhibited a 2-fold slower growth rate than wild type strains on glycerol or lactate. This growth phenotype is consistent with NAD+-dependent isocitrate dehydrogenase performing an essential role in the oxidative function of the citric acid cycle.
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